It is now recognized that the herpesvirus of horses, referred to as equine rhinopneumonitis (also called equine abortion virus, or EHV-1) is not a single herpesvirus but two genetically and antigenically distinct viruses, sometimes designated as subtypes 1 and 2 of EHV-1. EHV-1 subtype 1 (often called simple EHV-1) causes respiratory disease, spontaneous abortion in pregnant mares and occasionally, paralysis in horses. EHV-1 subtype 2 (also referred to as EHV-4) causes respiratory disease and only occasionally, abortions. The present invention is directed to a glycoprotein isolated from subtype l EHV-1 (hereafter referred to as EHV-1 in accordance with the International Committee on Taxonomy of Viruses at Edmonton, Canada in 1987).
Outbursts of EHV-1 infections in horses frequently occur in areas of concentrated horse breeding, particularly during the winter months. The incubation period of EHV-1 is from 2 to 10 days. Initial symptoms of infection include high fever for 1 to 7 days and discharge from the nostrils. White cell counts are generally depressed during the first few days of fever and may take a week or 10 days to recover. Diarrhea and enteritis, edema of the legs and tendovaginitis are not common in uncomplicated cases but do occur in complicated cases. All symptoms are worsened by forced exercise or work; recovery is complete in 1 to 2 weeks unless complications develop.
Reinfection may occur at intervals of 4 to 5 months or longer. These subsequent infections are usually asymptomatic and generally do not result in complications in adult horses. However, the disease has been known to breakout annually in young horses on farms where no new horses have been introduced, suggesting that adult horses can act as carriers. EHV-1 infection in young horses is often associated with weaning and assembling in winter quarter.
Infected mares may have no overt signs of infection at first, with the incubation time between nasal inoculation and abortion varying from 3 weeks to 4 months. The virus spreads readily by direct contact, fomites and aerosolized secretions. It may spread from one abortive mare to others, but evidence indicates that almost all mares on a farm are infected 1 to 4 months before abortion; hence, infection spreads rapidly, probably by aerosolized secretions or direct contact. Some foals infected prenatally reach full term and are born alive, but abortion is the normal outcome of EHV-1 infection in pregnant mares.
The herpesviruses are a family of structurally similar viruses. They have a double-stranded DNA genome characterized by short and long unique sequences of DNA (U.sub.s and U.sub.L respectively), and inverted repeats of DNA sequence which flank the unique sequences. The U.sub.s region of DNA is capable of inverting in orientation, giving rise to the prototype and inverted arrangements of the EHV-1 genome. All herpesviruses replicate within the nucleus of a host cell, and several members of the herpes family, if not all, are capable of becoming latent after establishing a primary infection and then initiating recurring, sometimes acute, infections.
Herpesviruses are not only similar in their gross morphology, but also at the molecular level. For example, general antisera against Herpes Simplex Virus type 1 (HSV-1) and EHV-1 have been used to demonstrate some minimal crossreaction between these viruses by complement fixation, gel diffusion, immunofluorescence and immunoprecipitation. However, HSV-1 has less than 5% DNA sequence identity with EHV-1 and specific antibodies to each virus do not cross-neutralize the other (Ludwig et al., 1971, Virology 45: 534-537). Despite this, the genome of EHV-1 appears to be functionally colinear with the genomes of HSV, pseudorabies virus (PRV) and varicella-zoster virus, as determined by molecular hybridization experiments (Davison et al., 1983, J. Gen. Virol. 64: 1927-1942). Analysis of the organization and function of the EHV-1 genome is therefore not only relevant for elucidating the mechanisms underlying EHV-1 infection, but also may identify key features of herpesvirus genomes by comparative molecular biology.
A number of major structural proteins have been identified in EHV-1 virions, typically by protein gel electrophoresis and through the use of antibodies directed against the intact EHV-1 virion. However, interest has centered on the structural glycoproteins due to their roles in the infectious process and their ability to invoke an immune response. In addition to several minor glycoproteins, eight high abundance glycoproteins have been identified in the envelope of purified EHV-1 virions. These glycoproteins have molecular masses of 200, 125, 95, 90, 68, 63, 45, and 41 kilodaltons (Perdue et al., 1974, Virology 59: 201-216; Turtinen et al., 1981, Am. J. Vet. Res. 42: 2099-2104), and are generally distinguished as glycoproteins by use of gp followed by the numbers 2, 10, 13, 14, 17, 18, 21, and 22a, respectively. Little is known about the antigenic or molecular structure of most of these glycoproteins. However the genes for six of these proteins have been mapped on the EHV-1 genome (gp2, gp10, gp13, gp14, gp17/18 and gp21/22a; Allen et al, 1987, J. Virol. 61: 2454-2461). All but gp 17/18 map within the long unique (U.sub.L) region of the EHV-1 genome.
Two of these six glycoproteins have been identified as homologs of glycoproteins known in other herpesviruses, based on map position: gp13 corresponds to gC of HSV (and gIII of PRY) and gp14 corresponds to gB of HSV (and gII of PRV) (Allen et al. 1987, supra). The nucleotide sequences of EHV-1 gp13 and gp14 have been determined and the translated amino acid sequences of both have revealed significant homology to the corresponding HSV glycoproteins (Allen et al., 1988, J. Virol 62: 2850-2858; Whalley et al., 1989, J. Gen. Virol. 70: 383-394). The HSV gB glycoprotein, with extensive amino acid sequence identity to EHV-1 gp14, is required for virus entry and cell fusion and has been shown to invoke circulating antibodies as well as cell-mediated immune response. Because of its structural similarity the gp14 protein may have a similar role.
A genomic library of EHV-1 DNA exists together with a physical, restriction map of the EHV-1 genome (Henry et al., 1981, Virology 115: 97-114). Identification and characterization of EHV-1 glycoproteins by analysis of the DNA in the unique short (U.sub.s) region of the EHV-1 genome has led to the discovery of a new EHV-1 glycoprotein (glycoprotein D).
There is a long standing need for safe, effective, long-acting, vaccines against. EHV-1 infection. A number of EHV-1 vaccines are currently available (e.g. U.S. Pat. No. 4,110,433 to Purdy; U.S. Pat. No. 4,083,958 to Bryans), but are derived from live viruses. In addition, the EHV-1 vaccines currently available are generally acknowledged as being inadequate in spectrum and duration of protection (Doll, 1961, J. Am. Vet. Med. Assoc. 139: 1324-1330; Bryans, 1976. In Equine Infectious Diseases IV, Proceedings of the Fourth International Conference on Equine Infectious Diseases, T. J. Bryans and H. Gerber, eds., Princeton: Veterinary Publications: 83-92; Burrows, et al., 1984, Veterin. Rec. 114: 369-374; and Stokes et al., 1989, J. Gen. Virol. 70: 1173-1183). Thus, the present discovery provides new vaccines for EHV-1 protection having significant advantages over those of the prior art, since the use of live or attenuated viruses is eliminated.